The present invention relates to new polypeptides involved in the biosynthesis of cobalamins and/or cobamides, and especially of coenzyme B12. It also relates to the genetic material responsible for the expression of these polypeptides, as well as to a method by means of which they may be prepared. It relates, lastly, to a method for amplification of the production of cobalamins, and more especially of coenzyme B12, by recombinant DNA techniques.
Vitamin B12 belongs to the B group of vitamins. It is a water-soluble vitamin which has been identified as the factor enabling patients suffering from pernicious anaemia to be treated. It is generally prescribed to stimulate haematopoiesis in fatigue subjects, but it is also used in many other cases comprising liver disorders and nervous deficiencies or as an appetite stimulant or an active principle with tonic activity, as well as in dermatology (Berck, 1982, Fraser et al., 1983). In the industrial rearing of non-ruminant animals, the feed being essentially based on proteins of vegetable origin, it is necessary to incorporate vitamin B12 in the feed rations in amounts of 10 to 15 mg per tonne of feed (Barrxc3xa8re et al., 1981).
Vitamin B12 belongs to a class of molecules known as cobalamins, the structure of which is presented in FIG. 1. Cobamides differ from cobalamins in the base of the lower nucleotide, which is no longer 5,6-dimethylbenzimidazole but another base, e.g. 5-hydroxybenzimidazole for vitamin B12-factor III synthesised, inter alia, by Clostridium thermoaceticum and Methanosarcina barkeri (Iron et al., 1984). These structural similarities explain the fact that the metabolic pathways of biosynthesis of cobalamins and cobamides are, for the most part, shared.
Cobalamins are synthesised almost exclusively by bacteria, according to a complex and still poorly understood process which may be divided into four steps (FIG. 2):
i) synthesis of uroporphyrinogen III (or uro""gen III), then
ii) conversion of uro""gen III to cobyrinic acid, followed by
iii) conversion of the latter to cobinamide, and
iv) construction of the lower nucleotide loop with incorporation of the particular base (5,6-dimethylbenzimidazole in the case of cobalamins).
For coenzyme B12, it is probable that the addition of the 5xe2x80x2-deoxyadenosyl group occurs shortly after the corrin ring-system is synthesised (Huennekens et al., 1982).
In the case of cobamides, only the step of synthesis and incorporation of the lower base is different.
The first part of the biosynthesis of cobalamins is very well known, since it is common to that of haemes as well as to that of chlorophylls (Battersby et al., 1980). It involves, successively, xcex4-aminolevulinate synthase (EC 2.3.137), xcex4-aminolevulinate dehydrase (EC 4.2.1.24), porphobilinogen deaminase (EC 4.3.1.8) and uro""gen III cosynthase (EC 4.2.1.75), which convert succinyl-CoA and glycine to uro""gen III. However, the first step takes place in some organisms [e.g. E. coli (Avissar et al., 1989) and in methanogenic bacteria (Kannangara et al., 1989), for example] by the conversion by means of a multi-enzyme complex of glutamic acid to xcex4-aminolevulinic acid.
Between uro""gen III and cobyrinic acid, only three intermediate derivatives have been purified to date; they are the factors FI, FII and FIII, which are oxidation products, respectively, of the three intermediates precorrin-1, precorrin-2 and precorrin-3, which correspond to the mono-, di- and trimethylated derivatives of uro""gen III (FIG. 3); these intermediates are obtained by successive transfers of methyl groups from SAM (S-adenosyl-L-methionine) to uro""gen III at positions C-2, C-7 and C-20, respectively. The other reactions which take place to give cobyrinic acid are, apart from five further transfers of methyl groups from SAM at C-17, C-12, C-1, C-15 and C-5, elimination of the carbon at C-20, decarboxylation at C-12 and insertion of a cobalt atom (FIG. 4). These biosynthetic steps have been deduced from experiments performed in vitro on acellular extracts of Propionibacterium shermanii or of Clostridium tetanomorphum. In these extracts, cobyrinic acid is obtained by conversion of uro""gen III after incubation under suitable anaerobic conditions (Batterby et al., 1982). No intermediate between precorrin-3 and cobyrinic acid capable of being converted to corrinoids by subsequent incubation with extracts of cobalamin-producing bacteria has been isolated to date. The difficulty of isolating and identifying these intermediates is linked to
i) their great instability,
ii) their sensitivity to oxygen, and
iii) their low level of accumulation in vivo.
In this part of the pathway, only one enzyme of Pseudomonas denitrificans has been purified and studied; it is SAM:uro""gen III methyltransferase (Blanche et al., 1989), referred to as SUMT.
Between cobyrinic acid and cobinamide, the following reactions are performed:
i) addition of the 5xe2x80x2-deoxyadenosyl group (if coenzyme B12 is the compound to be synthesised),
ii) amidation of six of the seven carboxyl functions by addition of amine groups, and
iii) amidation of the last carboxyl function (propionic acid chain of pyrrole ring D) by addition of (R)-1-amino-2-propanol (FIG. 2).
Whether there was really an order in the amidations was not elucidated (Herbert et al., 1970). Lastly, no assay of activity in this part of the pathway has been described, except as regards the addition of the 5xe2x80x2-deoxyadenosyl group (Huennekens et al., 1982).
The final step of the biosynthesis of a cobalamin, e.g. coenzyme B12, comprises four successive phases described in FIG. 5 (Huennekens et al., 1982), namely:
i) phosphorylation of the hydroxyl group of the aminopropanol residue of cobinamide to cobinamide phosphate, then
ii) addition of a guanosine diphosphate by reaction with guanosine 5xe2x80x2-triphosphate; the compound obtained is GDP-cobinamide (Friedmann, 1975), which
iii) reacts with 5,6-dimethylbenzimidazole, itself synthesised from riboflavin, to give adenosylcobalamin 5xe2x80x2-phosphate (Friedmann et al., 1968), which
iv) on dephosphorylation leads to coenzyme B12 (Schneider and Friedmann, 1972).
Among bacteria capable of producing cobalamins, the following may be mentioned in particular:
Agrobacterium tumefaciens 
Agrobacterium radiobacter 
Bacillus megaterium 
Clostridium sticklandii 
Clostridium tetanomorphum 
Clostridium thermoaceticum 
Corynebacterium XG 
Eubacterium limosum 
Methanobacterium arbophilicum 
Methanobacterium ivanovii 
Methanobacterium ruminantium 
Methanobacterium thermoautotrophicum 
Methanosarcina barkeri 
Propionobacterium shermanii 
Protaminobacter ruber 
Pseudomonas denitrificans 
Pseudomonas putida 
Rhizobium meliloti 
Rhodopseudomonas sphaeroides 
Salmonella typhimurium 
Spirulina platensis 
Streptomyces antibioticus 
Streptomyces aureofaciens 
Streptomyces griseus 
Streptomyces olivaceus 
At the industrial level, as a result of the great complexity of the biosynthetic mechanisms, the production of cobalamins, and especially of vitamin B12, is exclusively microbiological. It is carried out by large-volume cultures of the bacteria Pseudomonas denitrificans, Propionibacterium shermanii and Propionibacterium freudenreichii (Florent, 1986). The strains used for the industrial production are derived from wild-type strains; they may have undergone a large number of cycles of random mutation and then of selection of improved clones for the production of cobalamins (Florent, 1986). The mutations are obtained by mutagenesis with mutagenic agents or by physical treatments such as treatments with ultraviolet rays (Barrxc3xa8re et al., 1981). By this empirical method, random mutations are obtained and improve the production of cobalamins. For example, it is described that, from the original strain of Pseudomonas denitrificans initially isolated by Miller and Rosenblum (1960, U.S. Pat. No. 2,938,822), the production of this microorganism was gradually increased in the space of ten years, by the techniques mentioned above, from 0.6 mg/l to 60 mg/l (Florent, 1986). For bacteria of the genus Propionibacterium [Propionibacterium shermanii (ATCC 13673) and freudenreichii (ATCC 6207)], the same production values appear to be described in the literature; e.g. a production of 65 mg/l has been described (European Patent 87,920). However, no screen has yet been described enabling either mutants overproductive of cobalamins or mutants markedly improved in their production of cobalamins to be readily selected or identified.
At the genetic level, little work has been performed to date. The cloning of cob genes (coding for enzymes involved in the biosynthetic process) has been described in Bacillus megaterium (Brey et al., 1986). Eleven complementation groups have been identified by complementation of cob mutants of Bacillus megaterium with plasmids carrying different fragments of Bacillus megaterium DNA. These genes are grouped on the same locus, carried by a 12-kb fragment.
Studies have also been carried out on the cob genes of Salmonella typhimurium. Although the cloning of these has not been described, it has been shown that almost all the genes for cobalamin biosynthesis are grouped together between minutes 40 and 42 of the chromosome (Jeter and Roth, 1987). Only the cysG locus, which must permit the conversion of uro""gen III to precorrin-2, does not form part of this group of genes. However, the activity encoded by this locus and also its biochemical properties have not been described.
In addition, some phenotypes have been associated with cob mutations. In Salmonella typhimurium and in Bacillus megaterium, cob mutants no longer show growth on minimum medium with ethanolamine as a carbon source or as a nitrogen source (Roof and Roth, 1988). This is due to the fact that an enzyme of ethanolamine catabolism, ethanolamine ammonia-lyase (EC 4.3.1.7), has coenzyme B12 as a cofactor; the cob mutants no longer synthesise coenzyme B12, and they can no longer grow with ethanolamine as a carbon source and/or as a nitrogen source. metE mutants of Salmonella typhimurium retain only a methylcobalamin-dependent homocysteine methyltransferase (EC 2.1.1.13). cob mutants of Salmonella typhimurium metE are auxotrophic for methionine (Jeter et al., 1984).
In Pseudomonas denitrificans and Agrobacterium tumefaciens, phenotypes associated with a total deficiency of cobalamin synthesis have not been described to date.
Finally, work on Pseudomosas denitrificans (Cameron et al., 1989) has led to the cloning of DNA fragments carrying cob genes of this bacterium. These are distributed in four complementation groups carried by at least 30 kb of DNA. At least fourteen complementation groups have been identified by heterologous complementation of cob mutants of Agrobacterium tumefaciens and of Pseudomonas putida with DNA fragments of Pseudomonas denitrificans carrying cob genes.
However, hitherto, none of these genes has been purified, and no nucleotide sequence has been described. Similarly, no protein identification nor any catalytic function attributed to the product of these genes has been described. Furthermore, no improvement in production of cobalamins by recombinant DNA techniques could be obtained. The amplification of cob genes of Bacillus megaterium does not bring about, in the strain from which they have been cloned, an improvement in production of cobalamins (Brey et al., 1986). In Salmonella typhimurium, physiological studies have been carried out in order to determine conditions under which a strong transcription of the cob genes studied was observed (Escalante and Roth, 1987). Under these conditions, there is no improvement in the production of cobalamins, although genes of the biosynthetic pathway are more expressed than under standard culture conditions.
The present invention results from the precise identification of DNA sequences coding for polypeptides involved in the biosynthesis of cobalamins and/or cobamides. A subject of the invention hence relates to the DNA sequences coding for the polypeptides involved in the biosynthesis of cobalamines and/or cobamides. More especially, the subject of the invention is the cobA, cobB, cobC, cobD, cobE, cobF, cobG, cobH, cobI, cobJ, cobK, cobL, cobM, cobN, cobO, cobP, cobQ, cobS, cobT, cobU, cobV, cobW, cobX and corA genes, any DNA sequence homologous with these genes resulting from the degeneracy of the genetic code, and also DNA sequences, of any origin (natural, synthetic, recombinant), which hybridise and/or which display significant homologies with these sequences or with fragments of the latter, and which code for polypeptides involved in the biosynthesis of cobalamins and/or cobamides. The subject of the invention is also the genes containing these DNA sequences.
The DNA sequences according to the present invention were isolated from an industrial strain, Pseudomonas denitrificans SC510, derived from strain MB580 (U.S. Pat. No. 3,018,225), by complementation of cob mutants of A. tumefaciens and P. putida; and of Methanobacterium ivanovii. The clones obtained could be analysed precisely, in particular by mapping using insertions of a derivative of transposon Tn5. These genetic studies have enabled the cob or cor genes to be localised on the restriction map and their sequencing to be carried out. An analysis of the open reading frames then enabled the coding regions of these DNA fragments to be demonstrated.
The subject of the present invention is also the use of these nucleotide sequences for cloning the cob genes of other bacteria. In effect, it is known that, for proteins catalysing the same activities, sequences are conserved, the divergence being the evolutionary divergence (Wein-Hsiung et al., 1985). It is shown in the present invention that there is a significant homology between the nucleotide sequences of different microorganisms coding for polypeptides involved in the biosynthesis of cobalamins and/or cobamides. The differences which are seen result from the evolutionary degeneracy, and from the degeneracy of the genetic code which is linked to the percentage of GC in the genome of the microorganism studied (Wein-Hsiung et al., 1985).
According to the present invention, a probe may be made with one or more DNA sequences of Pseudomonas denitrificans in particular, or with fragments of these, or with similar sequences displaying a specific degree of degeneracy in respect of the use of the codons and the percentage of GC in the DNA of the bacterium which it is desired to study. Under these conditions, it is possible to detect a specific hybridisation signal between the probe and fragments of genomic DNA of the bacterium studied; this specific hybridisation signal corresponds to the hybridisation of the probe with the isofunctional cob genes of the bacterium. The cob genes as well as their products may then be isolated, purified and characterised. The invention thus provides a means enabling access to be gained, by hybridisation, to the nucleotide sequences and the polypeptides involved in the biosynthesis of cobalamins and/or cobamides of any microorganism.
The subject of the present invention is also a recombinant DNA containing at least one DNA sequence coding for a polypeptide involved in the biosynthesis of cobalamins and/or cobamides, and in particular a recombinant DNA in which the said sequence or sequences are placed under the control of expression signals.
In this connection, promoter regions may, in particular be positioned at the 5xe2x80x2 end of the DNA sequence. Such regions may be homologous or heterologous to the DNA sequence. In particular, strong bacterial promoters such as the promoter of the tryptophan operon Ptrp or of the lactose operon Plac of E. coli, the leftward or rightward promoter of bacteriophage lambda, the strong promoters of phages of bacteria such as Corynebacteria, the functional promoters in Gram-negative bacteria such as the Ptac promoter of E. coli, the PxylS promoter of the xylene catabolism genes of the TOL plasmid and the amylase promoter of Bacillus subtilis Pamy may be used. Promoters derived from glycolytic genes of yeasts may also be mentioned, such as the promoters of the genes coding for phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, lactase or enolase, which may be used when the recombinant DNA is to be introduced into a eukaryotic host. A ribosome binding site will also be positioned at the 5xe2x80x2 end of the DNA sequence, and it may be homologous or heterologous, such as the ribosome binding site of the cII gene of bacteriophage lambda.
Signals necessary to transcription termination may be placed at the 3xe2x80x2 end of the DNA sequence.
The recombinant DNA according to the present invention may then be introduced directly into a host cell compatible with the chosen expression signals, or be cloned into a plasmid vector to enable the DNA sequence in question to be introduced in a stable manner into the host cell.
Another subject of the invention relates to the plasmids thereby obtained, containing a DNA sequence coding for a polypeptide involved in the biosynthesis of cobalamins and/or cobamides. More specifically, these plasmids also contain a functional replication system and a selectable marker.
The subject of the invention is also the host cells into which one or more DNA sequences as defined above, or a plasmid as defined hereinbefore, has/have been introduced.
Another subject of the invention relates to a method for production of polypeptides involved in the biosynthesis of cobalamins and/or cobamides. According to this method, a host cell is transformed with a DNA sequence as described above, this transformed cell is cultured under conditions for expression of the said sequence and the polypeptides produced are then recovered.
The host cells which may be used for this purpose are either prokaryotes or eukaryotes, animal cells or plant cells. Preferably, they will be chosen from bacteria, and especially bacteria of the genus E. coli, P. denitrificans, A. tumefaciens or R. meliloti. 
Another use of the DNA sequences according to the present invention lies in a method for amplification of the production of cobalamins and/or cobamides, by recombinant DNA techniques. In effect, if the limitation of the metabolic flux of the biosynthesis of cobalamins and/or cobamides is due to a limitation in the activity of an enzyme in the biosynthetic pathway, an increase in this activity by increasing the expression of this same enzyme using recombinant DNA techniques (gene amplification, substitution of the transcription/translation signals with more effective signals, etc.) will lead to an increase in the biosynthesis of cobalamins and/or cobamides. It is also possible that the limitation of the production of cobalamins and/or cobamides results from a biochemical regulation. In this case, the cob gene or genes corresponding to the regulated enzyme may be specifically mutagenised in vitro in order to obtain mutated genes whose products will have lost the regulation mechanisms impeding an improvement in the production.
The method according to the present invention consists in transforming a microorganism productive of cobalamins and/or cobamides, or only potentially productive of these compounds (i.e. deficient in one or more steps of the biosynthesis), with a DNA sequence as defined above, then in culturing this microorganism under conditions for expression of the said sequence and for synthesis of cobalamins and/or cobamides, and lastly in recovering the cobalamins and/or cobamides produced. Such a method is applicable, in particular, to all the productive microorganisms mentioned on pages 5 and 6, and more specifically to microorganisms of the genus P. denitrificans, Rhizobium meliloti, or Agrobacterium tumefaciens. In a preferred embodiment, the microorganism is P. denitrificans, and especially strain SC510. As regards potentially productive microorganisms, the DNA sequences used will be those corresponding to the steps of the biosynthesis which the microorganism cannot carry out.
Using the present invention, and by the various stragegies described above, an improvement in the production of cobalamins and/or cobamides may be obtained for any microorganism productive or potentially productive of cobalamins and/or cobamides. It will suffice to culture this recombinant microorganism under suitable conditions for the production of cobalamins and for the expression of the DNA sequences introduced. This culturing may be carried out batchwise or alternatively in continuous fashion, and the purification of the cobalamins may be carried out by the methods already used industrially (Florent, 1986). These methods comprise, inter alia:
i) solubilisation of the cobalamins and their conversion to their cyano form (e.g. by heat treatment of the fermentation must, with potassium cyanide in the presence of sodium nitrite), then
ii) purification of the cyanocobalamins in various steps which can be, e.g.
a) adsorption on different substrates such as Amberlite IRC-50, Dowex 1xc3x972 or Amberlite XAD-2, followed by an elution with a water/alcohol or water/phenol mixture, then
b) extraction in an organic solvent, and lastly
c) precipitation or crystallisation from the organic phase, either by the addition of reagents or dilution in a suitable solvent, or by evaporation.
The present invention shows, furthermore, that it is possible by recombinant DNA techniques to improve the cobalamin production of a bacterium productive of cobalamins by cumulating improvements. This amounts to obtaining a first improvement as described above, and then in improving this improvement, still using recombinant DNA techniques, i.e., e.g. by amplifying genes for cobalamin biosynthesis.
Another subject of the present invention relates to the polypeptides involved in the biosynthesis of cobalamins and/or cobamides. In particular, the subject of the present invention is all polypeptides, or derivatives or fragments of these polypeptides, which are encoded by the DNA sequences described above, and which are involved in the pathway of biosynthesis of cobalamins and/or cobamides. The amino acid sequence of these polypeptides is described, as well as some of their physicochemical properties. An enzymatic activity or specific properties have also been associated with each of them.
In this connection, the subject of the invention is the polypeptides participating in the conversion of precorrin-3 to cobyrinic acid a,c-diamide, and more especially in the transfer of a methyl group from SAM to positions C-1, C-5, C-11, C-15 and C-17.
The subject of the invention is also the polypeptides:
participating in the conversion of cobyric acid to cobinamide, or
possessing an S-adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT) activity, or
possessing a cobyrinic and/or hydrogenobyrinic acid a,c-diamide synthase activity, or
possessing a precorrin-8x mutase activity, or
possessing a nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase activity, or
possessing a cobalamin-5xe2x80x2-phosphate synthase activity, or
possessing a cobyric acid synthase activity, or
possessing a cob(I)alamin adenosyl-transferase activity, or
possessing a precorrin-6x reductase activity, or
participating in the conversion of hydrogenobyrinic acid a,c-diamide to cobyrinic acid a,c-diamide.
Advantageously, the subject of the invention is a polypeptide chosen from the COBA, COBB, COBC, COBD, COBE, COBF, COBG, COBH, COBI, COBJ, COBK, COBL, COBM, COBN, COBO, COBP, COBQ, COBS, COBT, COBU, COBV, COBW, COBX and CORA proteins presented in FIGS. 15, 16, 40, 41 and 47.
Furthermore, the use of the hybridisation probes described above makes it possible, from genes isolated in other microorganisms, to characterise and isolate the isofunctional polypeptides of other microorganisms. In this manner, the present invention shows that the sequence of a COB protein of Pseudomonas denitrificans is significantly homologous with the protein sequences of other microorganisms displaying the same type of activity. Between these COB proteins catalysing the same reaction in different microorganisms, only the evolutionary distances have introduced variations (Wein-Hsiung et al., 1985). The subject of the present invention is also these isofunctional polypeptides.
The assignment of a particular enzymatic activity is the result of an analysis which may be performed according to various strategies. In particular, in vitro affinity studies with respect to SAM (S-adenosyl-L-methionine) make it possible to assign a methyl transferase activity to a protein capable of binding SAM, and hence to assign its involvement in one of the steps of transfer of methyl groups which occur between uro""gen III and cobyrinic acid. Another means of assessing the activity of these polypeptides consists in assaying the intermediates in the pathway of biosynthesis of cobalamins which are accumulated in mutants incapable of expressing these polypeptides (identified by complementation experiments). These analyses enable it to be deduced that the polypeptide in question has the accumulated intermediate as its substrate, thereby enabling its activity in the biosynthetic pathway to be situated and defined. The present invention also describes a method for assaying the enzymatic activities of the biosynthetic pathway, applicable to any strain productive of cobalamins and/or cobamides. These assays enable the enzymatic activity assayed to be purified from any strain productive of these compounds. From this purified activity, the NH2-terminal sequence of the COB protein in question, or alternatively that of the subunits of this protein, may be determined, thereby enabling the structural gene or genes which code for the activity in question to be identified. For Pseudomonas denitrificans, the structural genes which code for activities of the biosynthetic pathway are identified by finding, for each NH2-terminal sequence, the COB protein having the same NH2-terminal sequence.
The present invention also describes a method enabling intermediates in the pathway of biosynthesis of cobalamins or of other corrinoids to be identified and assayed in strains productive of cobalamins. These intermediates may be assayed both in culture musts and in the cells themselves. The intermediates which may be assayed are all the corrinoids which occur in the biosynthetic pathway after cobyrinic acid, namely, apart from cobyrinic acid, cobyrinic acid monoamide, cobyrinic acid diamide, cobyrinic acid triamide, cobyrinic acid tetraamide, cobyrinic acid pentaamide, cobyric acid, cobinamide, cobinamide phosphate, GDP-cobinamide, coenzyme B12 phosphate and coenzyme B12. The non-adenosylated forms of these products may also be assayed by this technique.
Other subjects and advantages of the present invention will become apparent on reading the examples and the drawings which follow, which are to be considered as illustrative and not limiting.
Definition of the Terms Employed and Abbreviations.